In the field of molecular diagnostics, the amplification and detection of nucleic acids is of considerable significance. Examples for diagnostic applications of nucleic acid amplification and detection are the detection of viruses such as Human Papilloma Virus (HPV), West Nile Virus (WNV) or the routine screening of blood donations for the presence of Human Immunodeficiency Virus (HIV), Hepatitis B (HBV) and/or C Virus (HCV) and the like. Furthermore, said amplification techniques are suitable for bacterial targets, or the analysis of oncology markers, or other targets.
Within a species, a microorganism or pathogen is often classified according to distinct groups, genotypes or subtypes based on nucleic acid sequence variation (i.e. HCV, HIV, HPV etc). In an in vitro diagnostic device, nevertheless, all groups, genotypes or subtypes should be detected and/or correctly quantified to avoid false negative diagnosis or wrong titer determination. This poses a considerable challenge for molecular diagnostic assays for e.g. detection of HIV and HCV.
Furthermore constant mutation and recombination of such pathogens generate within their target nucleic acids increasing diversity which must also be covered by the molecular diagnostic assay.
The most prominent and widely-used amplification technique is the Polymerase Chain Reaction (PCR). Other amplification reactions comprise, among others, the Ligase Chain Reaction, Polymerase Ligase Chain Reaction, Gap-LCR, Repair Chain Reaction, 3SR, NASBA, Strand Displacement Amplification (SDA), Transcription Mediated Amplification (TMA), and Qβ-amplification.
Automated systems for PCR-based analysis often make use of real-time detection of product amplification during the PCR process in the same reaction vessel. Key to such methods is the use of modified oligonucleotides carrying reporter groups or labels.
Detection of a microbial nucleic acid in a biological sample is crucial e.g. for recognizing an infection of an individual. Thereby, one important requirement e.g. for an assay for detection of a viral infection is inclusivity, defined such that false-negative results or underquantification of titers due to variable sequence regions on a viral genome caused by mutations have to be avoided. Mutated or partially mutated sequences within the respective genome that are possibly not amplified and/or detected in combination with the low viral load enhance the possibility of obtaining false-negative or falsely quantified results.
Several options have been published to increase the inclusivity of a molecular assay. Recently, coamplification of two different and non-overlapping target sequences within the genome of a pathogen was established (US 2010/0041040). This approach may, however, not be generally applicable if two reasonably conserved target regions cannot be identified within the genome of a pathogen or if the oligonucleotides for amplification and detection of two independent target regions interfere with each other in the master mix.
In this context, the prior art has e.g. provided methods for amplification and detection involving more than one probe based on homogeneous amplicon sequence with the aim to increase assay sensitivity (Yip et al., 2005, Clin. Chem. 51 (10)).